3.1 THE HUMAN IMMUNODEFICIENCY VIRUS
Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220: 868-870; Gallo et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). In humans, HIV replication occurs prominently in CD4.sup.+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed in part of capsid proteins designated p24 and p18, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammerskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV, like other enveloped viruses, introduces viral genetic material into the host cell through a viral-envelope mediated fusion of viral and target membranes. HIV is targeted to CD4.sup.+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (Pal et al., 1993, Virology 194:833-837; McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348), explaining HIV's tropism for CD4.sup.+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. The binding of gp120 to CD4 induces conformational changes in the viral glycoproteins, but this binding alone is insufficient to lead to infection (reviewed by Sattentau and Moore, 1993, Philos. Trans. R. Soc. London (Biol.) 342:59-66).
Studies of HIV-1 isolates have revealed a heterogeneity in their ability to infect different human cell types (reviewed by Miedema et al., 1994, Immunol. Rev. 140:35-72). The majority of extensively passaged laboratory strains of HIV-1 readily infect cultured T cell lines and primary T lymphocytes, but not primary monocytes or macrophages. These strains are termed T-tropic. T-tropic HIV-1 strains are more likely to be found in HIV-1 infected individuals during the late stages of AIDS (Weiss et al., 1996, Science 272:1885-1886). The majority of primary HIV-1 isolates (i.e., viruses not extensively passaged in culture) replicate efficiently in primary lymphocytes, monocytes and macrophages, but grow poorly in established T cell lines. These isolates have been termed M-tropic. The viral determinant of T- and M- tropism maps to alterations in the third variable region of gpl120 (the V3 loop)(Choe et al., 1996, Cell 85:1135-1148; Cheng-Mayer et al., 1991, J. Virol. 65:6931-6941; Hwang et al., 1991, Science 253:71-74; Kim et al., 1995, J. Virol., 69:1755-1761; and O'Brien et al., 1990, Nature 348:69-73). The characterization of HIV isolates with distinct tropisms taken together with the observation that binding to the CD4 cell surface protein alone is insufficient to lead to infection, suggest that cell-type specific cofactors might be required in addition to CD4 for HIV-1 entry into the host cell.
3.2 TREATMENT OF HIV INFECTION
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective. Therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H., et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2',3'-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H., et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddI, 3TC and ritonavir (Perelson, A. S., et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8.sup.+ T cells, which are essential to the control of HIV, via killer cell activity (Blazevic, V., et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of suppressive factors, notably the chemokines Rantes, MIP-1.alpha. and MIP-1.beta. (Cocchi, F., et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10:S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4.sup.+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4.sup.+ T cells by some HIV-1 strains (Smith, D.H., et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R., et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O., et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R., et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533).
Recently, chemokines produced by CD8.sup.+ T cells have been implicated in suppression of HIV infection (Paul, W. E., 1994, Cell 82:177; Bolognesi, D. P., 1993, Semin. Immunol. 5:203). The chemokines RANTES, MIP-1.alpha. and MIP-1.beta., which are secreted by CD8.sup.+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1 or HIV-2 isolates in vitro (Cocchi, F, et al., 1995, Science 270:1811-1815). Thus, these and other chemokines may prove useful in therapies for HIV infection. The clinical outcome, however, of all these and other candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L., et al., U.S. Pat. No. 5,141,867; Saith, G., et al., WO92/22,654; Shafferman, A., WO91/09,872; Formoso, C., et al., WO90/07,119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
3.3 WASTING SYDROMES
Wasting syndrome is a serious clinical problem characterized by a decrease in body mass of more than 10% from baseline body weight and a disproportionate loss of body mass with respect to body fat (Weinroth et al., 1995, Infectious Agents and Disease 4:76-94; Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275). Thus, wasting is distinguished from starvation in which higher levels of body fat than body cell mass are depleted (Kotler et al., 1985, Am. J. Clin. Nutr. 42:1255-1265; Cahill, 1970, N. Engl. J. Med. 282:668-675). Wasting is associated with a variety of conditions, including HIV infection, other infectious diseases, sepsis, cancer, chronic cardiovascular disease and diarrhea (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447; Heymsfield et al., 1982, Am. J. Clin. Nutr. 36:680-690). Importantly, wasting is a significant factor in the mortality of patients suffering from infections or cancer. In fact, body cell mass depletion has a linear relationship to time of survival in AIDS patients (Kotler et al., 1989, Am. J. Clin. Nutr. 50:444-447).
The cause of wasting syndrome in AIDS and other conditions is unclear and is most likely multifactorial. Metabolic abnormalities, irregular levels of hormones and cytokines, and malabsorption have all been implicated in wasting syndrome. Not all AIDS patients suffer from wasting, suggesting that the cause of the wasting is not HIV itself. Most cases of HIV associated wasting syndrome are apparently caused by complications of AIDS, such as secondary infections and gastrointestinal disease (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275).
Current and potential therapies for wasting syndromes include nutritional support, appetite enhancers such as dronabinol and megestrol acetate, anabolic therapies, such as growth hormone, and cytokine inhibitors. However, mixed results have been obtained with nutritional support and appetite enhancers in that patients tended to gain only fat and not overall body mass. Administration of growth hormone, and cytokine inhibitors are still being tested and may pose a risk of side effects (Kotler and Grunfeld, 1995, AIDS Clin. Rev. 96:229-275; Weinroth et al., 1995, Infectious Agents and Disease 4:76-94).
Thus, treatment of wasting is critical to the survival and well-being of patients suffering from serious diseases such as cancer and AIDS; thus, there is a need for safe and effective therapies for wasting syndrome associated with cancer, AIDS and other infectious diseases.
3.4 PROPERTIES OF DIMETHYLFORMAMIDE AND OTHER POLAR COMPOUNDS
N,N'-dimethylformamide (DMF) (molecular formula: C.sub.3 H.sub.7 ON) is a colorless, polar, hygroscopic liquid with low volatility and a boiling point of 152.5-153.5.degree. C. It is freely miscible with water, alcohols and some hydrocarbons. DMF is generally used as a polar solvent and is readily absorbed through the skin, by inhalation, and upon oral ingestion. DMF is rapidly metabolized, mainly in the liver, and excretion occurs principally in the urine. In rat, mouse, hamster and man the main metabolites of DMF are N-hydroxymethyl-N-methylformamide (HMMF), N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), as well as dihydroxymethylformamide (DHMF) and N-hydroxymethylformamide (HMF). Unchanged DMF is excreted in the urine as a small fraction of an administered dose of DMF.
DMF has low acute dermal, oral and inhalation toxicity. It is considered to be a mild to moderate skin and eye irritant and readily permeates the skin. There is no indication of skin sensitizing properties. The principal toxic effect of DMF and its metabolites is on the liver; DMF is well known to cause reversible hepatic damage associated with typical clinical complaints, classical biochemical changes in the blood, and the appearance of hepatocyte necrosis in liver biopsies. DMF is teratogenic, but is not thought to be a mutagen or a carcinogen.
Viza et al. have reported that DMF and DMSO inhibit in vitro replication of HIV and Human Herpes Virus 6 (HHV-6) in certain cultured cell lines. (See Viza et al., 1990, AIDS Res. Hum. Retroviruses 6:131-132; Viza et al., 1989, AIDS-FORSCHUNG 4:349-352; Viza et al., 1992, Antiviral Res. 18:27-38 and erratum at 19:179).
DMF has been described as an in vitro differentiating agent for certain transformed cells in culture (See Koeffler, 1983, Blood 62:709-721; Calabresi et al., 1979, Biochem. Pharmacol. 28:1933-1941). When added to certain malignant cells in vitro, DMF has been reported to reduce their tumorigenicity upon subsequent inoculation into nude mice (See Dexter, 1977, Cancer Res. 37:3136-3140; Dexter et al., 1979, Cancer Res. 39:1020-1025). Upon intraperitoneal injection into nude mice, DMF and NMF have been reported to slow the growth of certain human cancer xenografts (See Van Dongen et al., 1989, Int. J. Cancer 43:285-292; Braakhuis et al., 1989, Head & Neck 11:511-515; Van Dongen et al., 1988, Acta Otolaryngol. 105:488-493; Dexter et al., 1982, Cancer Res. 42:5018-5022). However, the toxic side-effects of formamide and its N-methyl derivatives in a mouse sarcoma allograft model led investigators to conclude that these agents were unlikely to prove therapeutically useful (See Clarke et al., 1953, Proc. Sec. Exp. Biol. Med. 84:203-207).
Attempts at treating human cancer patients with DMSO led to the conclusion that no objective response had been shown (See Spremulli & Dexter, 1984, J. Clin. Oncol. 2:227-241). Oral administration of NMF to human patients with cancer of the head and neck was reported as resulting in hepatotoxicity with no beneficial response (see Vogel et al., 1987, Invest. New Drugs 5:203-206), or with only minimal activity (See Planting et al., 1987, Cancer Treat Rep. 71:1293-1294).
U.S. Pat. No. 3,551,154 discloses the use of DMF as a penetration enhancer to promote transdermal absorption of topically applied medications. U.S. Pat. No. 4,855,294 discloses the use of glycerin to mitigate the skin irritation arising from the use of DMSO and DMF as penetration enhancers to promote transdermal absorption of topically applied medications. The use of DMSO as a penetration enhancer to promote transdermal absorption of antiviral agents is discussed in Woodford & Barry, 1986, J. Toxicol. Cut. & Ocular Toxicol. 5:167-177.
Citation or identification of any reference in Section 3 (or any other section) of this application shall not be construed as an admission that such reference is available as prior art to the present invention.